We are requesting funding to upgrade our Olympus FV1000 confocal microscope at the Optical Imaging Core (OIC) to a fully capable fluorescence lifetime (FLT) imaging microscope (FLIM) using system-specific upgrade kit from PicoQuant. FLIM measures FLT in cell microenvironment. Unlike fluorescence intensity, FLT is exquisitely sensitive to local probe environment through perturbation of excited state properties. Washington University in St. Louis (WUSTL) and surrounding research community do not have access to a FLIM system that provides easy-to-use interface and performs diverse wavelength imaging with the capability of detecting fluorescence in multiple channels. The FLIM system will support 17+ NIH-funded projects, initially consisting of 3 major and 11 minor users from WUSTL, Saint Louis University (SLU), and University of Missouri at St. Louis (UMSL). All the investigators have substantially published work in molecular imaging and demonstrable ongoing need for FLIM. Planned studies will focus on discovering novel molecular interactions that result in changes in the FLT of reporter molecules, designing new class of FLT contrast agents, quantitatively determining the sub-cellular distribution of target biomolecules, establishing the behavior of different fluorescent molecular probes and cancer drugs in cell compartments, understanding the complex dynamics of macromolecules in cells, unraveling the biochemical roles of proteins of unknown functions, and elucidating the bio-significance of FLT heterogeneity observed in noninvasive small animal imaging studies with high resolution FLIM. Not only will this system perform FLT and conventional fluorescence intensity measurement, it will also provide spatio-temporal information of a target biomolecule or process in response to cell microenvironmental changes stimulated by factors such as pH, lipophilicity, cell metabolic state, presence of free radicals, and hypoxia. The proposed system upgrade will enhance the services available in OIC for molecular imaging of biological processes and advance the research of many growing research groups at St. Louis. It will facilitate new collaborations and generate new concepts to advance our understanding of complex biological systems at cell level. Because of OIC's location in the heart of the medical center, the system will be available to all current and future users, as is the current practice with other instruments housed in OIC. Highly experienced personnel will manage the instrument, train new users, and help conduct new projects. The proximity of the instrument to research laboratories at WUSTL, SLU, and UMSL will attract new user and facilitate training of students and fellows using the state-of-the-art technology platform.